Positive detection lateral-flow apparatus and method for small and large analytes

ABSTRACT

Methods and devices for the detection and/or quantification of an analyte in a sample are provided. These are positive detection methods and devices, in that the more analyte is present in the sample, the stronger the signal that is provided. Devices of the invention include a mobilization zone including a mobile or mobilizable detectable analyte analog, a sample application area, primary and secondary capture areas each including an immobilized binding partner having a binding affinity for the analyte being tested for a detectable analyte analog. The mobilization zone, sample application area, primary and secondary capture area are in fluid continuous contact with each other. In these devices, the first immobilized binding partner has an equal or lower apparent affinity for the analyte than it has for the detectable analyte analog. Methods of this invention involve introducing a sample (which is suspected of containing the analyte to be tested for) to a device such as those described herein, and permitting the sample to migrate from the application area to and through the first and secondary binding zones. A detectable tracer conjugate is also permitted to migrate through the device, usually slightly behind the sample so that any analyte in the sample contacts the first binding partner before the conjugate. Results of such methods are read based on the presence and/or intensity of the detectable signal given by conjugate that binds in the second capture area.

[0001] This application claims priority from U.S. ProvisionalApplication No. 60/197,365 filed Apr. 14, 2000, and U.S. ProvisionalApplication No. 60/206,696 filed May 11, 2000, both of which areincorporated herein by reference.

FIELD

[0002] The present invention relates to sensitive lateral-flow methodsand devices for determining the presence and/or amount of small andlarge analytes in fluid samples. The present invention provides a director positive detection result (i.e. increasing signal with increasinganalyte concentration) in a sequential binding format.

BACKGROUND

[0003] Analytical tests have been developed for the routineidentification or monitoring of physiological and pathologicalconditions (e.g., pregnancy, cancer, endocrine disorders, infectiousdiseases) using different biological samples (e.g., urine, serum,plasma, blood, saliva), and for analysis of environmental samples (e.g.,natural fluids and industrial plant effluents) for instance forcontamination. Many of these tests are based on the highly specificinteractions between specific binding pairs. Examples of such bindingpairs include antigen/antibody, hapten/antibody, lectin/carbohydrate,apoprotein/cofactor and biotin/(strept)avidin. Furthermore, many ofthese tests involve devices (e.g., solid phase, lateral-flow teststrips, flowthrough tests) with one or more of the members of a bindingpair attached to a mobile or immobile solid phase material such as latexbeads, glass fibers, glass beads, cellulose strips or nitrocellulosemembranes (U.S. Pat. Nos. 4,703,017; 4,743,560; 5,073,484).

[0004] Immunochromatographic assays fall into two principal categories:“sandwich” and “competitive.” In general, sandwich immunochromatographicprocedures call for mixing the sample that may contain the analyte to beassayed with antibodies to the analyte. These antibodies are mobile andtypically are linked to a label or another signaling reagent, such asdyed latex, a colloidal metal sol, or a radioisotope. This mixture isthen applied to a chromatographic medium containing a band or zone ofimmobilized antibodies to the analyte of interest. The chromatographicmedium often is in the form of a strip that resembles a dipstick. Whenthe complex of the molecule to be assayed and the labeled antibodyreaches the zone of the immobilized antibodies on the chromatographicmedium, binding occurs and the bound, labeled antibodies are localizedat the zone. This indicates the presence of the molecule to be assayed.This technique can be used to obtain quantitative or semi-quantitativeresults. Examples of sandwich immunoassays performed on test strips aredescribed in U.S. Pat. Nos. 4,168,146 and 4,366,241, each of which isincorporated herein by reference.

[0005] In competitive immunoassays, the label is typically a labeledanalyte or analyte analogue that competes with any unlabeled analytepresent in the sample for binding to an antibody. In such competitiveassays, the analyte and labeled tracer molecule are simultaneouslyintroduced to the binding agent such that these molecules compete forbinding sites. Competitive immunoassays are typically used for detectionof analytes such as haptens, each hapten being monovalent and capable ofbinding only one antibody molecule. Examples of competitive immunoassaydevices are those disclosed by U.S. Pat. Nos. 4,235,601, 4,442,204 and5,208,535, each of which is incorporated herein by reference.

[0006] Solid phase immunoassay devices, whether sandwich- orcompetition-type, provide sensitive detection of an analyte in abiological fluid sample. Solid phase immunoassay devices incorporate asolid support to which one member of a ligand-receptor pair, usually anantibody, antigen, or hapten, is bound. Common early forms of solidsupports were plates, tubes, or beads of polystyrene, which were knownfrom the fields of radioimmunoassay and enzyme immunoassay. Morerecently, a number of porous materials such as nylon, nitrocellulose,cellulose acetate, glass fibers, and other porous polymers have beenemployed as solid supports.

[0007] In the more common forms of dipstick assays, as typified by homepregnancy and ovulation detection kits, immunochemical components suchas antibodies are bound to a solid phase. The assay device is “dipped”for incubation into a sample suspected of containing the subjectanalyte. Enzyme-labeled antibody is then added, either simultaneously orafter an incubation period. The device next is washed and then insertedinto a second solution containing a substrate for the enzyme. Theenzyme-label, if present, interacts with the substrate, causing theformation of colored products, which either deposit as a precipitateonto the solid phase or produce a visible color change in the substratesolution. EP-A 0 125 118 discloses such a sandwich type dipstickimmunoassay. EP-A 0 282 192 discloses a dipstick device for use incompetition type assays.

[0008] Flow-through type immunoassay devices (such as test strips) weredesigned to obviate the need for incubation and washing steps associatedwith dipstick assays. U.S. Pat. No. 4,632,901 discloses a sandwichimmunoassay device wherein antibody (specific to a target antigenanalyte) is bound to a porous membrane or filter to which a liquidsample is added. As the liquid flows through the membrane, targetanalyte binds to the antibody. The addition of sample is followed byaddition of labeled antibody. The visual detection of labeled antibodyprovides an indication of the presence of target antigen analyte in thesample.

[0009] Migration assay devices usually incorporate within them reagentsthat have been attached to colored labels, thereby permitting visibledetection of the assay results without addition of further substances.See, for example, U.S. Pat. No. 4,770,853; WO 88/08534; and EP-A 0 299428.

[0010] There are a number of commercially available lateral-flow typetests and patents disclosing methods for the detection of large analytes(MW greater than 1,000 Daltons). U.S. Pat. No. 5,229,073 describes asemiquantitative competitive immunoassay lateral flow method formeasuring plasma lipoprotein levels. This method utilizes a plurality ofcapture zones or lines containing immobilized antibodies to bind boththe labeled and free lipoprotein to give a semi-quantitative result.

[0011] U.S. Pat. No. 5,591,645 provides a chromatographic test stripwith at least two portions. The first portion includes a movable tracerand the second portion includes an immobilized binder capable of bindingto the analyte. Additional examples of lateral-flow tests for largeanalytes are disclosed in the following patent documents: U.S. Pat. Nos.4,168,146; 4,366,241; 4,855,240; 4,861,711; 5,120,643; European PatentNo. 0296724; WO 97/06439; and WO 98/36278.

[0012] There are also a limited number of lateral-flow type tests forthe detection of small-analytes (MW 100-1,000 Daltons). Generally, thesesmall analyte tests involve “typical” competitive inhibition to producenegative or indirect reporting results (i.e., reduction of signal withincreasing analyte concentration), as exemplified by U.S. Pat. No.4,703,017.

[0013] Several approaches have been developed for detecting smallanalytes using lateral-flow tests that produce positive or directreporting results (i.e., increase in signal with increasing analyteconcentration). These include, for instance, U.S. Pat. Nos. 5,451,504;5,451,507; 5,798,273; and 6,001,658.

[0014] U.S. Pat. No. 5,451,504 provides a method with three specificzones (mobilization, trap and detection) each containing a differentlatex conjugate to yield a positive signal. The mobilization zonecontains labeled antibody to bind the analyte in the sample. In the trapzone, unbound, labeled antibody is then trapped by immobilized analyteanalog. The detection zone captures the labeled analyte-antibodycomplex. A disadvantage of this method is that the analyte-analog in thetrap zone competes with the labeled analyte-antibody complex formedduring migration and may cause false negative results.

[0015] U.S. Pat. No. 5,451,507 describes a two-zone, disconnectedimmunochromatographic method. The first zone has non-diffusively boundreagent that binds with a component, e.g., an analyte analog bound to,or capable of becoming bound to, a member of a signal producing system.The second zone binds to the component only when the analyte to betested is present. The distance the component migrates into the secondzone is directly related to the concentration of analyte.

[0016] U.S. Pat. No. 5,798,273 discloses a lateral flow device thatincludes a capture zone with immobilized analyte analog and one or moreread-out zones to bind labeled analyte-analog. A disadvantage of thisdisclosed method is the requirement to premix sample, antibody andcolored label prior to application to the sample addition area of thelateral flow device.

[0017] U.S. Pat. No. 6,001,658 discloses a test strip device with adiffusible, labeled binding partner that binds with analyte, animmobilized analyte, and a detection area containing an immobilizedantibody.

[0018] A continuing need exists for a sensitive, rapid and single stepmethod to detect and quantify both large and small analytes at lowconcentrations (such as ng/ml or less).

SUMMARY

[0019] The present invention provides sensitive, rapid devices (in theform of a test strip) for determining the presence and/or amount (atng/ml levels) of small and/or large analytes in a fluid sample. Theinvention also provides methods for the determination of the presenceand/or amount of one or more components (e.g., analytes) in a sample.Results from the methods and devices disclosed herein can be positivelyread directly from the assay device by visual inspection or using anelectronic reader (such as a scanner).

[0020] The methods and devices disclosed herein can be used to detectanalytes in various types of fluid, including biological specimens (suchas blood, serum, plasma, urine, saliva, milk) and environmental samples(such as industrial plant effluent or natural fluids). Any known analytewith an appropriate analyte-specific antibody or other binding partnercan be easily detected and/or quantified using the disclosed methods anddevices. In certain examples of embodiments, a tracer is used, such ascolored or otherwise detectable particles (e.g., colored latex orcolloidal gold) conjugated to the analyte or an analyte-analog, whichtracers are collectively referred to as the conjugate.

[0021] Devices of the invention include a mobilization zone, a sampleapplication area, and primary and secondary capture areas. Each of thecapture areas includes an immobilized binding partner (such as anantibody) having a binding affinity for the analyte being tested and forthe detectable conjugate. A mobilization zone, sample application area,and primary and secondary capture areas are in fluid continuous contactwith each other, e.g., on a lateral flow chromatography strip. In thesedevices, the first immobilized binding partner binds the analyte anddetectable conjugate. However, to the extent that analyte is present inthe sample, the labeled conjugate is less able or unable to bind in theprimary capture zone and instead continues to migrate along the striptoward the secondary capture zone, where it binds with the secondarybinding partner. The labeled conjugate therefore provides a positivesignal, in proportion to the amount of analyte present in the sample, inthe secondary capture zone.

[0022] In some embodiments, the specific binding agent in the primarycapture zone may have a higher affinity for the analyte than the labeledconjugate, such that the conjugate is less preferentially bound in theprimary capture zone and preferentially passes through to the secondarycapture zone. However, the binding agent in the primary capture zone mayhave an equal affinity for the analyte, and the labeled analyte willstill pass through the primary capture zone to bind in the secondarycapture zone and emit a signal proportional to the presence of analytein the sample. It is also possible that the binding agent in the primarycapture zone may have a lower affinity for the analyte in someembodiments, and still provide a substantially quantitative signal fromthe secondary capture zone, with the signal proportional to the amountof analyte in the sample.

[0023] Other embodiments of the test strip permit sequential migrationof the analyte and labeled analyte analog, such that the analytemigrates in advance of the labeled conjugate, to be bound by thespecific binding agent in the primary capture zone. Since sites in theprimary capture zone are already occupied when the labeled conjugatereaches the primary capture zone, the conjugate continues to migratealong the strip to the secondary capture zone. The labeled conjugatethen provides a signal in the secondary capture zone, which signal isproportionate to the amount of analyte in the sample.

[0024] Certain methods of this invention involve introducing a liquidsample (which is suspected of containing the analyte to be tested for)on to the test strip, and permitting the sample to migrate along thetest strip by capillary action from the application area to and throughthe first and secondary binding zones. The tracer conjugate may bepresent in the application zone, in the path of migration, or appliedseparately to the strip. The conjugate also migrates along the teststrip, for example slightly behind the sample, so that any analyte inthe sample contacts the first binding partner before the analyte-tracerconjugate. If the analyte is present in the sample, it will bind to thefirst binding partner, and occupy binding sites that are then notavailable to bind the labeled conjugate. Hence the conjugate willmigrate along the strip to the secondary binding zone, where thepresence and/or intensity of the detectable signal positively indicatesan amount of analyte in the sample.

[0025] The foregoing and other features and advantages of the inventionwill become more apparent from the following detailed description ofseveral embodiments, which proceeds with reference to the accompanyingFigures.

BRIEF DESCRIPTION OF THE FIGURES

[0026]FIG. 1A is a top plan view of a first embodiment of the lateralflow test strip of the present invention.

[0027]FIG. 1B is a side view of the test strip shown in FIG. 1A.

[0028] FIGS. 1C-4F are schematic top views of the test strip of FIG. 1A,illustrating the progressive advancement of a liquid specimen bycapillary action through the strip.

[0029]FIG. 2A is a top plan view of a second embodiment of the teststrip.

[0030]FIG. 2B is a side view of the test strip shown in FIG. 2A.

[0031] FIGS. 2C-2F are schematic top views of the test strip of FIG. 2A,illustrating the progressive advancement of a liquid specimen throughthe strip.

[0032]FIG. 3A is a top plan view of a third embodiment of the teststrip.

[0033]FIG. 3B is a side view of the test strip shown in FIG. 3A FIGS.3C-3F are schematic tops views of the test strip of FIG. 3A,illustrating the progressive advancement of a liquid specimen throughthe strip.

[0034]FIG. 4 is a schematic view of a series of test strips to whichhave been applied different concentrations of an analyte, whichillustrates how quantitative information about the concentration of theanalyte can be obtained from the intensity of test bands shown on thestrip.

[0035]FIG. 5 is a graph that illustrates a substantially linearrelationship between a color reaction on the test strip and a cotinineanalyte concentration in a saliva test specimen.

[0036]FIG. 6 is a schematic view similar to FIG. 4, but showing a stripwith four indicator lines, which quantitatively indicate an amount ofanalyte in the sample.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS

[0037] I. Abbreviations and Definitions

[0038] A. Abbreviations

[0039] CDTA: trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid

[0040] EDTA: ethylenediamine tetraacetate

[0041] EGTA: ethyleneglycol-bis(β-oxyethylenenitrilo)-tetraacetic acid

[0042] Ig: immunoglobulin

[0043] IGEPAL: IGEPAL GA-630 (octylphenoxy)polyethoxyethanol

[0044] GAM: Goat-anti-mouse antibody

[0045] MAb: monoclonal antibody

[0046] NTA: nitriloacetic acid

[0047] PVA: polyvinyl alcohol PVP: p0 polyvinylpyrrolidone

[0048] B. Definitions

[0049] Analyte—an atom, molecule, group of molecules or compound ofnatural or synthetic origin (e.g. drug, hormone, enzyme, growth factorantigen, antibody, hapten, lectin, apoprotein, cofactor) sought to bedetected or measured that is capable of binding specifically to at leastone binding partner (e.g. drug, hormone, antigen, antibody, hapten,lectin, apoprotein, cofactor).

[0050] The methods of this invention can be practiced with assays forvirtually any analyte. The analytes may include, but are not limited toantibodies to infectious agents (such as HIV, HTLV, Helicobacter pylori,hepatitis, measles, mumps, or rubella), cocaine, benzoylecgonine,benzodizazpine, tetrahydrocannabinol, nicotine, ethanol theophylline,phenytoin, acetaminophen, lithium, diazepam, nortryptyline,secobarbital, phenobarbitol, methamphetamine, theophylline,testosterone, estradiol, estriol, 17-hydroxyprogesterone, progesterone,thyroxine, thyroid stimulating hormone, follicle stimulating hormone,luteinizing hormone, transforming growth factor alpha, epidermal growthfactor, insulin-like growth factor I and II, growth hormone releaseinhibiting factor, IGA and sex hormone binding globulin; and otheranalytes including antibiotics (e.g., penicillin), glucose, cholesterol,caffeine, cotinine, corticosteroid binding globulin, PSA, or DHEAbinding glycoprotein.

[0051] Analytes vary in size. Merely by way of example, small moleculeanalytes may be, for instance, <0.1 nm (such as cotinine or penicillin,each with a molecular weight of less than about 1,000 Daltons). However,analytes may be larger than this, including for instance immunoglobulinanalytes (such as IgG, which is about 8 nm in length and about 160,000Daltons).

[0052] Analyte analog—a modified analyte that has structural similarityto the unmodified analyte and can bind to at least one analyte bindingpartner. In certain embodiments of the invention, the analyte analog isan analyte-tracer conjugate, for instance a detectable analyte-tracerconjugate.

[0053] Antibody—a protein consisting of one or more polypeptidessubstantially encoded by immunoglobulin genes or fragments ofimmunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0054] The basic immunoglobulin (antibody) structural unit is generallya tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and “variable heavy chain” (V_(H)) refer, respectively, to theselight and heavy chains.

[0055] Antibodies may exist as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)——C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab withpart of the hinge region (see, Fundamental Immunology, W. E. Paul, ed.,Raven Press, N.Y., 1993). While various antibody fragments are definedin terms of the digestion of an intact antibody, it will be appreciatedthat Fab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies.

[0056] Antibodies for use in the methods and devices of the inventioncan be monoclonal or polyclonal, but often will be monoclonal. Merely byway of example, such monoclonal antibodies can be prepared from murinehybridomas according to the classical method of Kohler and Milstein(Nature 256:495-497, 1975) or derivative methods thereof. Briefly, amouse is repetitively inoculated with a few micrograms of the selectedanalyte compound (or a fragment thereof) over a period of a few weeks.In some instances, it will be beneficial to use an adjuvant or a carriermolecule to increase the immunogenicity and/or stability of the analytein the animal system. The mouse is then sacrificed, and theantibody-producing cells of the spleen isolated. The spleen cells arefused by means of polyethylene glycol with mouse myeloma cells, and theexcess un-fused cells destroyed by growth of the system on selectivemedia comprising aminopterin (HAT media). The successfully fused cellsare diluted and aliquots of the dilution placed in wells of a microtiterplate where growth of the culture is continued. Antibody-producingclones are identified by detection of antibody in the supernatant fluidof the wells by immunoassay procedures, such as ELISA, as originallydescribed by Engvall (Meth. Enzymol. 70:419-439, 1980), and derivativemethods thereof. Selected positive clones can be expanded and theirmonoclonal antibody product harvested for use. Detailed procedures formonoclonal antibody production are described in Harlow and Lane(Antibodies, A Laboratory Manual, CSHL, New York, 1988).

[0057] Monoclonal antibodies to different analytes are commerciallyavailable. For instance, a monoclonal antibody to estriol-3 is producedby Fitzgerald Industries International (Concord, Mass.; Cat. #10-E37,Clone #M612039); likewise, Omega Biological, Inc. (Bozeman, Mont.)produces a monoclonal antibody to methamphetamine (Cat. #100-11-183,Clone Met 2).

[0058] Antigenic—a chemical or biochemical structure, determinant,antigen or portion thereof that is capable of inducing the formation ofan antibody.

[0059] Binding affinity—a term that refers to the strength of binding ofone molecule to another at a site on the molecule. If a particularmolecule will bind to or specifically associate with another particularmolecule, these two molecules are said to exhibit binding affinity foreach other. Binding affinity is related to the association constant anddissociation constant for a pair of molecules, but it is not critical tothe invention that these constants be measured or determined. Rather,affinities as used herein to describe interactions between molecules ofthe described methods and devices are generally apparent affinities(unless otherwise specified) observed in empirical studies, which can beused to compare the relative strength with which one molecule (e.g., anantibody or other specific binding partner) will bind two othermolecules (e.g., an analyte and an analyte-tracer conjugate). Theconcepts of binding affinity, association constant, and dissociationconstant are well known.

[0060] In the devices and methods of this invention, the immobilizedspecific binding partners do not generally have a lower binding affinityfor the analyte-tracer conjugate than they do for the analyte beingtested for in a sample. Rather, the immobilized binding partners willhave the same apparent affinity for the analyte and analyte-tracerconjugate, or in some instances will have greater affinity for theanalyte-tracer conjugate. In certain embodiments, an immobilized bindingpartner may have little or no affinity for the analyte while havingstrong or very strong affinity for the analyte tracer conjugate (forinstance, this will be the case for some immobilized binding partnersthat are immobilized in the secondary capture zone).

[0061] Binding domain—the molecular structure associated with thatportion of a receptor that binds ligand. More particularly, the bindingdomain may refer to a polypeptide, natural or synthetic, or nucleic acidencoding such a polypeptide, the amino acid sequence of which representsa specific (binding domain) region of a protein, which either alone orin combination with other domains, exhibits binding characteristics thatare the same or similar to those of a desired ligand/receptor bindingpair. Neither the specific sequences nor the specific boundaries of suchdomains are critical, so long as binding activity is exhibited.Likewise, used in this context, binding characteristics necessarilyincludes a range of affinities, avidities and specificities, andcombinations thereof, so long as binding activity is exhibited.

[0062] Binding partner—any molecule or composition capable ofrecognizing and binding to a specific structural aspect of anothermolecule or composition. Examples of such binding partners andcorresponding molecule or composition include antigen/antibody,hapten/antibody, lectin/carbohydrate, apoprotein/cofactor andbiotin/(strept)avidin.

[0063] Chelator—(chelating resin) a composition that binds divalentcations. The binding can be reversible or irreversible. Binding ofdivalent cations generally renders them substantially unable toparticipate in chemical reactions with other moieties with which theycome in contact. Chelators are well known and include ethylenediaminetetraacetate (EDTA), sodium citrate,ethyleneglycol-bis(β-oxyethylenenitrilo)-tetraacetic acid (EGTA),trans-1,2-diaminocyclohexaneN,N,N′,N′-tetraacetic acid (CDTA),nitriloacetic acid (NTA), resins that contain moieties that binddivalent cations and the like. Chelators that remain in solid phase inthe solution in question are referred to as chelating resins. Chelatingresins can be used to pull the subject ion (e.g., Ca²⁺) out of solution.Chelating resins include, but are not limited to, chelex resinscontaining iminodiacetate ions, resins containing free base polyamines,aminophosphonic acid, and the like.

[0064] Immunogen—a chemical or biochemical structure, determinant,antigen or portion thereof, that elicits an immune response, including,for example, polylysine, bovine serum albumin and keyhole limpethemocyanin (KLH).

[0065] Label—any molecule or composition bound to an analyte, analyte,analog or binding partner that is detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Examples of labels, including enzymes, colloidal goldparticles, colored latex particles, have been disclosed (U.S. Pat. Nos.4,275,149; 4,313,734; 4,373,932; and 4,954,452, each incorporated byreference herein).

[0066] The attachment of a compound (e.g., an analyte) to a label can bethrough covalent bonds, adsorption processes, hydrophobic and/orelectrostatic bonds, as in chelates and the like, or combinations ofthese bonds and interactions and/or may involve a linking group.

[0067] Lateral flow device: Devices that include bibulous ornon-bibulous matrices capable of transporting analytes and reagents to apre-selected site. Many such devices are known, in which the strips aremade of nitrocellulose, paper, cellulose, and other bibulous materials.Non-bibulous materials can be used, and rendered bibulous by applying asurfactant to the material.

[0068] Lateral flow chromatography strip: A test strip used in lateralflow chromatography, in which a test sample fluid, suspected ofcontaining an analyte, flows (for example by capillary action) throughthe strip (which is frequently made of materials such as paper ornitrocellulose). The test fluid and any suspended analyte can flow alongthe strip to a detection zone in which the analyte (if present)interacts with a detection agent to indicate a presence, absence and/orquantity of the analyte.

[0069] Linking group—a chemical arm between two compounds, for instancea compound and a label (e.g., an analyte and a label). To accomplish therequisite chemical structure, each of the reactants must contain areactive group. Representative combinations of such groups are aminowith carboxyl to form amide linkages; carboxy with hydroxy to form esterlinkages or amino with alkyl halides to form alkylamino linkages; thiolswith thiols to form disulfides; or thiols with maleimides oralkylhalides to form thioethers. Hydroxyl, carboxyl, amino and otherfunctionalities, where not present in the native compound may beintroduced by known methods.

[0070] Likewise, a wide variety of linking groups may be employed. Thestructure of the linkage should be a stable covalent linkage formed toattach two compounds to each other (e.g., the label to the analyte). Insome cases the linking group may be designed to be either hydrophilic orhydrophobic in order to enhance the desired binding characteristics, forinstance of the modified ligand and its cognate receptor. The covalentlinkages should be stable relative to the solution conditions to whichlinked compounds are subjected. Examples of linking groups will be from1-20 carbons and 0-10 heteroatoms (NH, O, S) and may be branched orstraight chain. Without limiting the foregoing, it should be obviousthat only combinations of atoms that are chemically compatible comprisethe linking group. For example, amide, ester, thioether, thiol ester,keto, hydroxyl, carboxyl, ether groups in combinations withcarbon-carbon bonds are particular examples of chemically compatiblelinking groups.

[0071] Operable or contiguous contact—two solid components are inoperable contact when they are in contact, either directly orindirectly, in such a manner that an aqueous liquid can flow from one ofthe two components to the other substantially uninterruptedly, bycapillarity or otherwise. Direct or contiguous contact means that thetwo elements are in physical contact, such as edge-to-edge orfront-to-back. When two components are in direct contact, they mayoverlap with an overlap of about 0.5 to about 3 mm. However, thecomponents can be placed with abutting edges. “Indirect contact” meansthat the two elements are not in physical contact, but are bridged byone or more conductors. Operable contact can also be referred to as“fluid transmitting” or “fluid continuous” contact.

[0072] Positive/direct reporting—an increase in the reporting ordetection signal with increasing analyte concentration.

[0073] Preservative—a substance showing antimicrobial properties, inparticular bactericidal properties and, in some instances alsoantifungal properties.

[0074] Specific binding partner—a member of a pair of molecules thatinteract by means of specific, noncovalent interactions that depend onthe three-dimensional structures of the molecules involved. Typicalpairs of specific binding partners include antigen/antibody,hapten/antibody, hormone/receptor, nucleic acid strand/complementarynucleic acid strand, substrate/enzyme, inhibitor/enzyme,carbohydrate/lectin, biotin/(strept)avidin, and virus/cellular receptor.

[0075] The phrase “specifically binds to an analyte” or “specificallyimmunoreactive with,” when referring to an antibody, refers to a bindingreaction which is determinative of the presence of the analyte in thepresence of a heterogeneous population of molecules such as proteins andother biologic molecules. Thus, under designated immunoassay conditions,the specified antibodies bind to a particular analyte and do not bind ina significant amount to other analytes present in the sample. A varietyof immunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular analyte. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow and Lane,Antibodies, A Laboratory Manual, CSHP, New York (1988), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity.

[0076] II. Detailed Description of the Figures

[0077] FIGS. 1-3 show three different embodiments of a lateral flowchromatography strip of the present invention, which will beindividually described.

Embodiment of FIG. 1

[0078] The lateral flow chromatography strip 20 is seen in isolation inFIGS. 1A and 1B, which show strip 20 to include an elongated, narrow,bibulous liquid collection member 22 with a flat proximal edge 24 and aflat distal edge 26. Strip 20 is mounted on a rigid or semi-rigidplastic support 28, and a proximal absorbent sample collection pad 30 isalso mounted to the support 28 such that it is contiguous withcollection member 22, and includes a reduced thickness extension 32 thatextends in a distal direction over member 22. A distal reservoir pad 34is attached to a distal end of support 28, and reservoir pad 34 includesa reduced thickness extension 36 that overlaps subjacent collectionmember 22. Liquid placed on collection pad 30 moves by capillary actionin a distal direction 38 through collection member 22 into reservoir pad34.

[0079] Capture agents (such as specific binding partners, for exampleantibodies such as monoclonal antibodies) are aligned in spacedindicator lines 50, 52, each of which extends transversely on the strip.A mobilization zone 54 is located on collection member 22 between pad 30and indicator line 50. The mobilization zone 54 contains an analyte (oranalyte analog) linked to a tag, hereinafter referred to as an A-L-Tconjugate. In the embodiment illustrated in FIG. 1, the analyte-tracerconjugate is dried in mobilization zone 54 on strip 20. In thisembodiment, the fluid sample, applied to sample pad 30 (FIG. 1C) andmoves through pad 30 and overlapping extension 32. The sample thenmigrates by capillary action through the porous material of collectionmember 22 (FIG. 1D). As the fluid sample migrates through the porousmaterial, it mobilizes the analyte-tracer conjugate A-L-T, which thenflows with the liquid sample through the primary capture line 50 (FIG.1E) and secondary capture line 52 (FIG. 1F), into reservoir 34. As shownin FIGS. 1E and 1F, the presence of analyte in the sample is detected bya positive signal (a color change) in secondary capture line 52.

[0080] The illustrated embodiment incorporates different monoclonalantibodies in each indicator line, and the monoclonal antibodies of theprimary and secondary capture zones have different affinities for theanalyte and conjugate. In a specifically illustrated embodiment, theprimary monoclonal antibodies of the primary capture zone are all thesame, and have an equal or greater affinity for the analyte than theconjugate. The monoclonal antibodies are attached to the substrate in aknown fashion, and binding of conjugate to these antibodies provides isdetectable (such as by a change in color, electrical conductivity,fluorescence, or magnetic polarity) if the labeled conjugate binds tothe capture agent. The secondary capture area serves as the readoutarea, indicating the test result. The test results can be visualizeddirectly, or may be measured using a reader device (e.g., a scanner).Such reader devices may detect color or fluorescence from the readoutarea.

[0081] The sample pad 30 may be a pad of polyester, glass fiber, orcellulose, to which the fluid sample is applied. The collection member22 may, for example, be a porous material such as nitrocellulose. Thereservoir pad 34, for absorbing excess fluid that flows through thecollection member 22, is composed for example of cellulose or anyfluid-absorbent material. The backing support 28 may be a plastic suchas polyvinylchloride (PVC) or any other liquid-impervious material. Theantibodies or binding partners that are immobilized in the primary andsecondary capture lines 50, 52 may be the same or different, and theantibodies or binding partners in each capture area may be immobilizedin single or multiple (distinct) lines (although only a single line 50and single line 52 are shown in FIG. 1). Primary capture line 50 canalso serve as a control area to indicate that the device is functioningproperly, since the presence of signal from primary capture line 50indicates that the conjugate is present, and that the capture agentbinds the conjugate.

Embodiment of FIG. 2 A-L-T Conjugate Mobilization Zone Under Sample Pad

[0082] Another embodiment of a lateral flow chromatography strip 120 isseen in isolation in FIGS. 2A and 2B, which is similar to FIGS. 1A and1B, and wherein like parts have been given like reference numbers plus100. FIGS. 2A and 2B show strip 120 to include an elongated, narrow,bibulous liquid collection member 122 with a flat proximal edge 124 anda flat distal edge 126. Strip 120 is mounted on a rigid or semi-rigidplastic support 128, and a proximal absorbent sample collection pad 130is also mounted to the support 128 such that it is contiguous withcollection member 122, and includes a reduced thickness extension 132that extends in a distal direction over member 122. A distal reservoirpad 134 is attached to a distal end of support 128, and reservoir pad134 includes a reduced thickness extension 136 that overlaps subjacentcollection member 122. Liquid placed on collection pad 130 moves bycapillary action in a distal direction 138 through collection member 122into reservoir pad 134.

[0083] Capture agents (such as specific binding partners, for exampleantibodies such as monoclonal antibodies) are aligned in spacedindicator lines 150, 152, each of which extends transversely on thestrip, and respectively form the primary and secondary capture zones. Amobilization zone 154 is located on collection member 122 underneath pad130 and indicator line 150. The mobilization zone 154 contains ananalyte (or analyte analog) linked to a tag (and referred to as an A-L-Tconjugate). In the embodiment illustrated in FIG. 2, the analyte-tracerconjugate is dried in mobilization zone 154 on strip 120 prior to pad132 being applied to support 128. In this embodiment, the fluid sampleis applied to sample pad 130 (FIG. 2C), which mobilizes the A-L-Tconjugate in mobilization zone 154. The A-L-T conjugate moves with theliquid sample through pad 130 and overlapping extension 132 (FIG. 2D).The A-L-T conjugate can also be dried in mobilization zone 154 on pad132 before pad 132 is applied to support 128.

[0084] Since the sample is applied to the surface of pad 130, it isbelieved that it encounters less resistance and migrates more quicklythrough pad 130 than the subjacent A-L-T conjugate (which must behydrated and mobilized), and it is believed that the sample (and anyanalyte in the sample) therefore reaches the primary capture line 150(FIG. 2E) before the A-L-T conjugate. Once the A-L-T conjugate reachesthe antibodies in primary capture line 150, the specific binding sitesare already occupied by any analyte from the sample, which reduces thenumber of binding sites available to bind A-L-T. Hence the A-L-Tconjugate continues to migrate by capillary action through the porousmaterial of collection member 122 until it reaches secondary captureline 152 (FIG. 2F), where it is bound by the specific binding partnerimmobilized therein. As shown in FIGS. 2E and 2F, the presence ofanalyte in the sample is detected by a positive signal (a color change)in secondary capture line 152.

[0085] The illustrated embodiment incorporates different monoclonalantibodies in each indicator line, and the monoclonal antibodies of theprimary and secondary capture zones have the same or differentaffinities for the analyte and conjugate. In a specifically illustratedembodiment, the primary monoclonal antibodies of the primary capturezone are all the same, and the secondary monoclonal antibodies of thesecondary capture zone are all the same. However, the primary monoclonalantibodies have an equal or greater affinity for the analyte than theconjugate. In the embodiment in which the primary antibody has a greateraffinity for the analyte than the conjugate, the primary antibodiespreferentially bind the analyte, which further inhibits binding of theA-L-T conjugate in primary capture line 150. The unbound A-L-T thereforemore readily moves through primary capture line 150 to bind at secondarycapture line 152.

[0086] As with the embodiment of FIG. 1, the secondary capture line 152provides an indication (such as a change in color, electricalconductivity, fluorescence, or magnetic polarity) if the labeled A-L-Tconjugate binds to the capture agent.

Embodiment of FIG. 3 Separate Introduction of A-L-T Conjugate on toStrip

[0087] A third embodiment of a lateral flow chromatography strip 220 isseen in FIGS. 3A and 3B, which is similar to FIGS. 1A and 1B, andwherein like parts have been given like reference numbers plus 200. Inthis embodiment, the A-L-T conjugate is not present on the strip itself,but is instead applied separately to strip 220 after the sample has beenplaced on the strip, so that the sample (and any analyte containedtherein) will reach the primary and secondary capture lines 250, 252before the A-L-T conjugate.

[0088]FIGS. 3A and 3B show strip 220 to include an elongated, narrow,bibulous liquid collection member 222 with a flat proximal edge 224 anda flat distal edge 226. Strip 220 is mounted on a rigid or semi-rigidplastic support 228, and a proximal absorbent sample collection pad 230is also mounted to the support 228 such that it is contiguous withcollection member 222, and includes a reduced thickness extension 232that extends in a distal direction over member 222. A distal reservoirpad 234 is attached to a distal end of support 228, and reservoir pad234 includes a reduced thickness extension 236 that overlaps subjacentcollection member 222. Liquid placed on collection pad 230 moves bycapillary action in a distal direction 238 through collection member 222into reservoir pad 234.

[0089] In this third embodiment, the A-L-T conjugate is applied inliquid form to the sample application pad 230 (FIG. 3D) during or soonafter application of the sample to pad 230 (FIG. 3C). If the A-L-Tconjugate is applied after application of the sample to pad 230, and atsubstantially the same place on pad 230, then the sample will reachcapture lines 250, 252 prior to the A-L-T conjugate reaching the capturelines. Alternatively, the sample and A-L-T conjugate may be appliedsubstantially simultaneously to pad 230 (not shown), but the sample isapplied at a more distal position on strip 220 (closer to primarycapture line 250). The sample and analyte-tracer conjugate then migrateat substantially a same rate by capillary action through the porousmaterial of strip 220. However, the sample (and any analyte therein)reaches primary capture line 250 before the A-L-T conjugate, such thatany analyte binds to the specific capture agents in primary capture line250, and occupies binding sites that are unavailable for binding of theA-L-T conjugate to capture line 250. Hence the unbound A-L-T conjugatecontinues to migrate through the strip to secondary capture line 252,where it is bound and provides a signal of its presence (whichcorrelates to the presence and amount of analyte in the test sample).

[0090] If analyte is not present in the sample, binding of A-L-Tconjugate occurs at primary capture line 250, and all (or substantiallyall) of the A-L-T conjugate is bound at primary line 250, so that asignal is absent or weak at secondary line 252. Moreover, since thesignal at secondary line 252 is proportional to the quantity of A-L-Tconjugate that is not bound by primary line 250, and which migrates tosecondary line 252, the intensity of the signal from secondary line 252is proportional to a concentration of analyte in the sample.

[0091] In another embodiment, the sample and A-L-T conjugate can bedesigned to migrate at different rates through the porous strip, toallow any analyte in the sample to reach the primary capture line beforethe A-L-T conjugate. For example, the A-L-T conjugate can be a verylarge molecule that migrates more slowly than the free analyte in thesample. For example, a bovine serum albumin is a very large moleculethat can be used as a linker L between the analyte analog A and tracerT, which can increase the size and molecular weight of the A-L-Tconjugate relative to the free analyte. Hence, the smaller free analytewill migrate more quickly through the strip than the large A-L-Tconjugate. For instance, the tracer molecule can be selected (based onsize, polarity, charge, or other such characteristics) to providespecific migration characteristics.

[0092] By way of example only, colloidal gold particles can be as smallas about 20 nm, but may be larger. In specific examples whereincolloidal gold particles are used as the tracer element of an A-L-T ofthis invention, the particles will be about 30 nm, about 40 nm, about 50nm, or about 60 nm or larger, depending in part on the migrationcharacteristics desired. Likewise, in embodiments that incorporate latexparticles (e.g., colored latex particles) as the tracer element, latexparticles can be about 200 nm, about 250 nm, about 300 nm, about 350 nm,or about 400 nm or larger. However, when large tracer elements are used,it is important that the diameter of the entire A-L-T complex be takeninto account. It is possible that, if the diameter of the entire complexis greater than the typical pore size of the porous flow material, thecomplex (or a portion thereof) may be trapped and not migrate. Inaddition, large A-L-T complexes might in some instances create sterichindrance that reduces the interaction between the analyte or analyteanalog and the immobilized binding partner. In each instance, however,with the provision of the methods herein, empirical testing ofindividual, specific A-L-T complexes, as well as combinations of A-L-Tand the particular immobilized binding partner(s) and porous flowmaterial(s), is enabled.

[0093] In these three illustrated embodiments, the fluid migrates alongthe porous material of the strip by capillary action into the reservoirpad. Once the reservoir pad becomes saturated, the fluid migrationautomatically stops.

[0094] In these three illustrated embodiments, the specific analyte andanalyte-tracer conjugate particles sequentially “compete” for binding tothe specific binding partner localized at the primary capture area, inthat the analyte (if any is present) first contacts the binding partnerin the primary area and the conjugate (which follows after) can onlybind to specific binding partners that remain unoccupied. In the casewhere a sample does not contain the specific analyte, the analyte-tracerconjugate particles bind with the binding partner in the primary capturezone. Minimal or no analyte-tracer particle is then available for travelto and binding with the immobilized binding partner in the secondarycapture area, resulting in no signal. In the case where a samplecontains the specific analyte to be detected, the binding partner in theprimary capture area binds free analyte in the sample and there arefewer (if any) primary capture binding sites available for binding tothe analyte-tracer complex. The portion of analyte-tracer complex thatfails to bind to binding sites in the first capture area (due toprevious binding by the analyte) will be captured by the immobilizedbinding partner in the secondary capture “read-out” area. As appliedsample contains increasing amounts of target analyte, greater amounts ofanalyte-tracer particles (unbound in the primary capture area) are freeto bind to the secondary capture area, resulting in an increased signal.

[0095] III. Positive Detection Lateral Flow Devices and Methods

[0096] Devices according to the invention generally include a strip ofabsorbent material, which can be made of different substances eachjoined to the other in zones, which may be abutted and/or overlapped.The absorbent strips are fixed on a solid support. Zones within eachstrip may differentially contain the specific binding partner(s) and/orother reagents required for the detection and/or quantification of theparticular analyte being tested for. Thus these zones can be viewed asfunctional sectors or functional regions within the test device. Ingeneral, a fluid sample (or a sample suspended in a fluid) is introducedto the strip at the proximal end of the strip, for instance by dippingor spotting. The fluid migrates distally through all the functionalregions of the strip. The final distribution of the fluid in theindividual functional regions depends on the adsorptive capacity and thedimensions of the materials used. Within these bounds, substantialvariation exists within the several examples of the devices of thisinvention, as will be explained below.

[0097] The described methods and devices can be used to detect any smallor large analyte having at least one binding-specific epitope thatpermits the binding of a specific binding partner. This invention relieson the binding of one binding partner to a single epitope on theanalyte, rather than the binding of two binding partners (each of whichbinds to a different epitope on the analyte) as in used in classic“sandwich” format immunochromatographic tests; therefore the disclosedmethods and devices can detect analytes smaller than about 1,000Daltons. Such small analytes generally do not have two epitopes, andthus cannot be detected using a sandwich format assay.

[0098] A. Construction and Configuration

[0099] In some embodiments, the sample application pad (such as pad 32in FIGS. 1A and 1B) can have the function of a volume-metering element,as described in German Patent Nos. 3,043,608 and 2,332,760, and U.S.Pat. Nos. 3,464,560, 3,600,306, 3,667,607, 3,902,847, 4,144,306 and4,258,001. Examples of the sample application pad are a piece of fabricor paper which is located at one end of the test element and whichbecomes completely saturated with a volume (for instance, a definite ormeasured volume) of liquid merely by being dipped into a fluid, forexample a solution of the sample, and then releases the liquid to thesucceeding zones more slowly and in a controlled manner. The pad hasdimensions such that it takes up sufficient fluid to permit the fluid tomigrate to the other end of the device, to the reservoir pad orabsorption zone (such as pad 34 in FIGS. 1A and 1B). The absorption padhas the function of absorbing excess and freely mobile reagentcomponents and reaction products of the signal-producing system.

[0100] The absorbent materials that form the strip of the discloseddevices can, for instance, be composed of cellulose; chemicalderivatives of cellulose or of plastics having a porous or fibrousstructure and adequately hydrophilic properties; particles such ascellulose or silica gel embedded in a synthetic membrane; and also ofnatural products that are hydrophilic but have been rendered insolublein water. A combination of strips composed of different materials can beused. Suitable absorbent materials are selected on the basis of therequirements set for the particular assay device. Nitrocellulose,commonly used to bind and immobilize proteins and polyethylene glycols,is one example of a material for use in making the collection member ofthe test strips of this invention. Polysulfones, nylons or other porousmembranes capable of adsorbing macromolecules also provide examples ofstrip material for use with devices and methods of this invention.

[0101] The liquid impervious backing, to which the sample applicationpad, porous (absorbent) material strip(s), and reservoir pad areattached, may be polyvinylchloride (PVC) or any other liquid-imperviousmaterial.

[0102] Test strips of the invention will usually be arranged linearly,with the sample application zone (area) being at the proximal end of thestrip, and the reservoir pad (adsorption zone) being at the distal endof the strip. Between these two ends will be located the primary andsecondary capture zones, with the primary capture zone located closer tothe application zone and the secondary capture zone located closer tothe reservoir pad. The mobilizable analyte-tracer conjugate (A-L-T) canbe located beneath the application zone (covered by the sample pad), ordistal to the application zone (e.g., between the application zone andthe primary capture zone). Mobile analyte-tracer conjugate (which isadded to the test strip at the same time as or after application of thesample) can be placed at any point along the test strip so long as it isproximal to the primary capture zone.

[0103] Individual components of the devices of this invention arearranged in such a way that the sample suspected of containing analytecontacts the primary (first) capture zone before the analyte tracerconjugate does. This permits the analyte (if present) to bind to theavailable binding sites first, followed by the binding of analyte-tracerconjugate to any remaining vacant specific binding sites in the firstcapture area. The delay in the analyte-tracer conjugate contact with thefirst capture area can be achieved by the arrangement of theanalyte-tracer conjugate on the test strip (for instance by placing theanalyte-tracer conjugate distal to the sample application area), byadding the analyte-tracer conjugate to the test strip at the same timeor after application of the sample, or by providing delayed-releasereversible immobilization of the analyte-tracer conjugate to the surfaceof the test device strip. Such delayed-release can be accomplished, forexample, through application of the A-L-T complex to the test stripmaterial in the presence of one or more co-molecules, such as sucrose(about 5-50%), mannitol (about 5-30%); glycerol (about 1-15%), PVA(about 0.1-5%), PVP (about 0.1-5%), or mixtures thereof.

[0104] Certain embodiments of the current invention will include atleast one filter element, which is placed at the sample application zoneor between the sample application zone and the primary capture line.Such a filter element may increase the sensitivity of the assay byremoving unwanted components in the fluid sample and yet allowingunimpeded passage of labeled analyte. Thus, a proportionately greateramount of analyte binds to the assay read-out area, and more accurateassay results are achieved.

[0105] B. Choice of Analyte

[0106] The assay device of this invention can detect small analytes, forinstance those of interest in medical diagnostics. Small analytes can,for instance, be nutrients, peptides, hormones (e.g., human chorionicgonadotropin (hCG), frequently assayed as a marker of human pregnancy,estrogen or progesterone), drugs (both therapeutic drugs such asantibiotics, tranquilizers and anticonvulsants, and drugs of abuse suchas cocaine, heroin, and marijuana), environmental pollutants (e.g.,pesticides and aromatic hydrocarbons), and vitamins, as well asderivatives, metabolites (catabolites or anabolites), fragments oranalogs of these molecules. One specific example of a small analyte iscotinine, a major metabolite of nicotine, detection of which can be usedfor determining the smoking status of an individual.

[0107] Larger analytes detectable using the methods and devicesdisclosed herein can be antigens (for instance, antigens specific tobacterial, viral, and protozoan pathogens, such as Streptococcus,hepatitis virus, and Giardia), antibodies (such as antibodies induced asa result of infection with pathogens, such as antibodies to thebacterium Helicobacter pylori and to human immunodeficiency virus),enzymes (such as aspartate aminotransferase, lactate dehydrogenase,alkaline phosphatase, glutamate dehydrogenase, and other indicators ofphysiological function and/or tissue damage), and other proteins (suchas hemoglobin, frequently assayed in determinations of fecal occultblood, an early indicator of gastrointestinal disorders such as coloncancer).

[0108] C. Choice of Binding Partner(s)

[0109] The binding partner(s) (e.g., antibodies) immobilized on theporous material of the test device may be applied directly or through acarrier molecule such as protein A, protein G, or anti-immunoglobulin.One common technique used to immobilize protein-derived binding partners(e.g., antibodies) onto nitrocellulose or another solid support is toirreversibly adsorb them onto the solid support. Methods for attachingthe binding partner(s) to such carrier molecules are well known, and canfor instance involve the use of one or more linking groups.

[0110] The diagnostic devices and methods described in the presentinvention can be used in any binding pair reactions, for instance thosereactions having immunochemical components such as antibodies, antigens,and haptens. In those assays in which the detection of analyte moleculesis described herein, the molecules immobilized in the assay indicatorzones are analyte binding molecules. In some examples, both immobilizedreagents will be anti-analyte antibodies. The antibodies can be eithermonoclonal or polyclonal, the methods of producing which are well knownin the art. Any combination of monoclonal-polyclonal antibodies can beemployed.

[0111] The immobilized specific binding partner in the primary capturezone will have an affinity for the analyte being tested for, and anaffinity for the analyte-tracer conjugate being used as a reportingmolecule. When the first binding partner does not have a lower bindingaffinity (has an equal or greater binding affinity) for theanalyte-tracer conjugate than it has for the analyte, the analyte-tracerconjugate is at least as likely to bind to the immobilized bindingpartner in the first binding zone as will a molecule of the analytebeing tested for. In some embodiments, the first binding partner willhave a higher affinity for the analyte-tracer than it has for theanalyte, but it will have at least some affinity for the analyte. Theabsolute values of these binding affinities, however, are not essentialto the invention; rather, it is the relative affinity of the analyte andthe analyte-tracer conjugate for the first immobilized specific bindingpartner that is important. A lower or equal affinity for theanalyte-tracer conjugate helps increase the likelihood that thismolecule, traversing the first capture area after the analyte has doneso, does not dislodge a significant amount of the analyte from theimmobilized binding partner molecules.

[0112] The secondary capture zone can also contain one or more distinctlines of immobilized antibody or binding partner. This secondimmobilized binding partner can but need not be the same as theimmobilized binding partner in the first binding zone. In embodimentswhere the first and second immobilized binding partners are different,the second binding partner may have a high binding affinity for theanalyte-tracer complex and will have the same, less or no bindingaffinity to free analyte that is being measured. In particularembodiments, this second immobilized binding partner will not have ahigher binding affinity for the analyte than it does for theanalyte-tracer conjugate.

[0113] The purpose of the second capture area is to trap theanalyte-tracer conjugate that was not trapped in the first capture area,thereby generating a signal that can be read as the result of the test.

[0114] D. Choice of Ligand/Analog/Analyte-Tracer Conjugate

[0115] The invention includes the use of an analyte-tracer conjugate.The analyte-tracer conjugate includes an analyte or analyte-analog, suchthat the conjugate shares or mimics at least one three-dimensionaldeterminant(s) of the native analyte to be detected. In sharing at leastone binding determinant with the analyte target, the analyte-tracerconjugate can bind to at least certain specific binding partners thatbind the analyte. The analyte tracer conjugate also includes some sortof reporter molecule, for example a visible colored or fluorescentparticle such as a polystyrene latex microsphere, or colloidal gold. Thetracer can be attached to the analyte (or analyte analog) by a linkinggroup. Other examples of reporter (tracer) molecule are an enzyme,fluorophore, or other molecule known to produce a detectable and/ormeasurable product or signal.

[0116] The analyte-tracer conjugate generally includes at least onelabel that makes the amount of the bound analog detectable and/orrecordable by measuring techniques. Labels can be, for example:radioactive isotopes, enzymes, fluorescent, phosphorescent orluminescent substances, substances having stable unpaired electrons,erythrocytes, latex particles (including dyed latex such as described inWO 88/08534, EP-A 0 280 559 and 0 281 327), magnetic particles, metalsols (such as gold sol particles such as those described in U.S. Pat.No. 4,313,734), dye sol particles such as described in U.S. Pat. No.4,373,932 and WO 88/08534, and dyes encapsulated in liposomes, asdescribed in U.S. Pat. No. 4,703,017.

[0117] In some examples, the analyte analog (analyte-tracer conjugate)will be bound to or otherwise associated with another, non-labelingmolecule. This carrier molecule can function, for instance, to attachthe label (tracer) to the analyte analog (as discussed above), and/ormay provide additional stability or longevity to the analyte-tracerconjugate. Examples of such carrier molecules are bovine serum albumin(BSA), polyethylene glycol (PEG) (the use of which is described inhereby incorporated U.S. Pat. No. 6,033,918), avidin or streptavidin,and immunoglobulins.

[0118] E. Choice and Preparation of Sample

[0119] The choice of the sample will in part be governed by the analyteto be detected. It will be obvious that the sample should be chosen tobe one in which it is suspected the analyte may be present. Medicallyrelevant substances (e.g., analytes) can be found in blood (includingantibodies, antigens, drugs, hormones, enzymes, metabolites, peptidesand so forth), tears, sweat, and other secretions and exudates.

[0120] For instance, many substances (e.g., analytes) of medicalsignificance can be found in saliva (Malamud and Tabak (eds.), Saliva asa Diagnostic Fluid, Ann. NY Acad. Sci., 1993, ISBN #08 976 67883). Theseinclude drugs (e.g., illegal or abused drugs such as marijuana oralcohol, as described in U.S. Pat. No. 5,968,746), drug metabolites(such as cotinine, a bi-product of nicotine metabolism), hormones (suchas estriol, which is linked to risk of pre-term labor (McGregor et al.,Am. J. Obstet. Gynecol., 173:1337-1342, 1995)), electrolytes (such asthose useful in monitoring ovulation, as described in U.S. Pat. No.5,914,271), antibiotics, infectious agents (e.g., through the presenceof antibodies to the infectious agents, as described for instance inU.S. Pat. No. 5,942,403), and so forth.

[0121] The use of saliva samples for medical tests is now well known, asare methods for the collection, preparation (such as filtration orpretreatment of the sample, see below), and preservation (see forinstance U.S. Pat. No. 5,968,746) of saliva samples. Saliva can becollected using, for instance, a sponge, an absorbent pad, asalt-impregnated absorbent pad, chewable substrates, an aspirator (e.g.,a straw, capillary tube, or syringe), a mouth rinse, or byexpectoration. Specialized devices or mechanisms also can be used tocollect saliva samples (see, e.g., U.S. Pat. Nos. 6,022,326 and5,871,905). Certain devices for collection of oral fluids arecommercially available, for instance, from Epitope, Inc., Beaverton,Oreg., or Saliva Diagnostic Systems, Vancouver, Wash.

[0122] It will be beneficial with certain samples to remove at least aportion of any particulates that may be in the sample. Such particulates(depending on the sample) may include blood cells, other cellulardebris, food or other oral particles, and/or sediment (e.g., fromnatural water samples). Such particulates can be removed usingtechniques that are well known, including filtration and sedimentation.

[0123] As disclosed in U.S. Pat. No. 5,871,905, adding mixtures of bilesalts (or their corresponding acid forms) and, optionally, agents whichchelate or sequester divalent ions to saliva or other oral samples mayreduce the incidence of false positives sometimes seen with suchsamples. For examples of the disclosed devices and methods, the bileacid or salt (such as deoxycholic acid (deoxycholate salt), cholic acid(cholate salt), chenodeoxycholic acid (chenodeoxycholate salt),glycodeoxycholic acid (glycodeoxycholate salt), and/or taurodeoxycholicacid (taurodeoxycholate salt)) is present in a concentration sufficientto reduce the rate of occurrence of false positives in the assay.Examples of chelators include, but are not limited to, EDTA, EGTA, NTA,CDTA, sodium citrate, and a chelating resin. For instance, EDTAconcentrations in the final sample used in the assay can range fromabout 0.005 M to about 0.05 M.

[0124] IV. Kits for Detection and/or Quantification of Analyte

[0125] Assay devices according to the invention can be provided in theform of kits. Such kits will include one or more assay devices (whichmay be for the same or different analytes), and instructions for the useof the device(s). The instructions may provide direction on how to applysample to the test device, how to apply analyte-tracer conjugate inthose kits wherein the device does not have conjugate dried thereon, theamount of time necessary or advisable to wait for results to develop,and details on how to read and interpret the results of the test. Suchinstructions may also include standards, such as standard tables,graphs, or pictures for comparison of the results of a test. Thesestandards may optionally include the information necessary to quantifyanalyte using the test device, such as a standard curve relatingintensity of signal or number of signal lines to an amount of analytetherefore present in the sample.

[0126] In those embodiments wherein the analyte-tracer conjugate isadded to the test device after or during application of the sample, thekit will also include an aliquot of the analyte-tracer analog. Suchaliquot can be provided in solubilized or solubilizable form, and willbe provided in a container. In certain kits, the amount ofanalyte-tracer conjugate provided will be sufficient for use with asingle assay device, where as other kits (particularly those whichcontain more than one assay device) will contain sufficientanalyte-tracer compound to perform several tests.

EXAMPLES

[0127] The following examples are provided to illustrate certainparticular features of the present invention. These examples should notbe construed to limit the invention to the particular featuresdescribed.

Example 1 Cotinine Test with Liquid Analyte Tracer Conjugate Reagents

[0128] A bovine serum albumin (BSA)-cotinine conjugate was preparedusing cotinine trans-4-carboxylic acid as follows: To 0.25 ml of amixture of dimethylformamide and pyridine (1/1 by volume) was added 5 mgof trans-4-carboxycotinine (Sigma-Aldrich, St. Louis, Mo.), 5 mg ofN-hydroxysuccinimide and 15 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Sigma-Aldrich, St. Louis, Mo.). The mixturewas rotated to dissolve, and then left rotating for 1 h to form theactive ester as described previously (Fjrowell and Landon, Ann. Clin.Biochem. 23: 596-602, 1986). A 50 μl aliquot of this active ester wasadded to 1 ml of 10 mg/ml of BSA solution in 0.1 M phosphate buffer (pH7.4). The reaction mixture was rotated at room temperature for 4 hours,then dialyzed against 10 mM phosphate buffer (pH 7.4). The BSA-cotinineconjugate was diluted to a concentration of 5 mg/ml bovine serum albuminin 10 mM phosphate buffer (pH 7.4) and stored.

[0129] The BSA-cotinine conjugate was coupled to carboxyl blue latexparticles using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to formthe cotinine-tracer conjugate as follows: To 1.0 ml of 10 mg/ml ofcarboxyl latex particles (0.33 μm, Bangs Laboratories, Fishers IN) in0.1 M phosphate buffer (pH 7.0) was added 10 mg of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The mixture was shakenfor 20 minutes at room temperature. The activated latex particles werecentrifuged and then suspended in 1 ml of 1 mg /ml of BSA-cotinineconjugate (5 mg/ml BSA-cotinine conjugate diluted 1:5 in 0.1 M boratebuffer, pH 8.0). The reaction mixture was rotated at room temperaturefor 3 hours. To this reaction mixture was added 10 μl of 10% BSA (indistilled water) to block the unreacted sites. The resultingcotinine-tracer conjugate was washed twice with 10 mM phosphate buffer(pH 7.4) containing 0.1% BSA, 1 mM EDTA and 0.05% Tween 20. Thecotinine-tracer conjugate was suspended to a concentration of 10 mg/mllatex in the same phosphate buffer and stored.

[0130] Anti-cotinine antibody (MAb1) was purified from tissue culturemedium by protein A agarose chromatography (see Example 6, below).

[0131] Anti-cotinine-BSA antibody (MAb2) was purified from tissueculture medium by protein A agarose chromatography (see Example 6,below).

[0132] Goat-anti mouse IgG (GAM) antibody was purified from serum byaffinity chromatography using standard techniques.

[0133] Cotinine (Sigma-Aldrich, St. Louis, Mo.) test solutions (10, 50,100 and 200 ng/ml) were prepared in a saliva matrix (75 mM phosphatebuffered saline, pH 7.4, 2.5 mM EDTA, 0.05% Triton X100, 0.05% IGEPAL,0.1% sodium benzoate, 0.1% potassium sorbate, 6 mM potassium chloride,10 mM potassium carbonate, 6 mM sodium chloride, 5 mM sodium phosphate,0.1 mM magnesium chloride, 1 mM calcium chloride, and 50 μg/ml BSA, 500μg/ml mucin).

Construction of Test Strips

[0134] A 2.4×30 cm strip of nitrocellulose was affixed to a 15 miladhesive coated vinyl backing.

[0135] MAb1 was mixed (1:1) with GAM to form a stable soluble complex.One line of a 1.0 mg/ml solution of the MAb1-GAM complex was applied to(sprayed on) the nitrocellulose membrane (capture/control line) toproduce a total applied complex of 1 μg/linear centimeter. One line of a500 μg/ml solution of MAb2 was applied to (sprayed on) thenitrocellulose membrane (test/read line) to produce a total appliedcomplex of 0.5 μg/linear centimeter. The nitrocellulose membrane wasdried for 10 minutes at 37° C., then blocked with a stabilized buffer(StabilGuard™, Surmodics Inc., Eden Prairie, Minn.) diluted with anequal volume of 150 mM phosphate buffered saline (pH 7.4, .05% sodiumazide), and dried again for 30 minutes at 37° C.

[0136] A polyester pad was wetted by immersion in a 1.0% Triton X-100solution in 100 mM phosphate buffer at pH 7.4, then laminated onto thelower portion of the strip to act as a sample wick. A cellulose pad waslaminated to the upper portion of the strip to act as absorbentreservoir.

[0137] The completed card was cut into 4 mm strips. Strips were insertedinto plastic housings with a hole/well located in the sample applicationarea and a window in the test reading area.

Test Procedure

[0138] A 20 μl aliquot of cotinine test solution was added to the samplewell of each test strip device and allowed to absorb for 10-30 seconds.Thereafter, 20 μl of cotinine-tracer conjugate was added to the samplewell of each test strip device. The test strips were allowed to developfor 5-10 minutes.

Results

[0139] The test strips were read visually. The results are depicted inFIG. 4 and summarized below:

[0140] 10 ng cotinine/ml: control line visible, no test line visible;

[0141] 50 ng cotinine/ml: control line visible, weak test line visible;

[0142] 100 ng cotinine/ml: control line visible, strong test linevisible; and

[0143] 200 ng cotinine/ml: control line visible, very strong test linevisible.

[0144] The color intensity of the test line on each test strip was readwith a Minolta CR241 calorimeter. The direct relationship between thecolor intensity reading and cotinine concentration in the saliva matrixis summarized in FIG. 5.

Example 2 Cotinine Test With Multiple Read Lines Reagents

[0145] A BSA-cotinine conjugate prepared as in Example 1 was coupled tocarboxyl blue latex particles using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as described in Example 1.

[0146] Anti-cotinine monoclonal antibody (MAb1) as described in Example1 was diluted to 1 mg/ml in PBS (pH 7.4, 0.5% azide).

[0147] Goat-anti mouse IgG antibody (GAM) was purified from serum byaffinity chromatography using standard techniques.

[0148] Cotinine (Sigma-Aldrich, St. Louis, Mo.) test solutions (0, 20,200, 500 ng/ml) were prepared in saliva matrix as described above.

Test Strips

[0149] A 2.4×30 cm strip of nitrocellulose was affixed to a 15 miladhesive coated vinyl backing. MAb1 solution was mixed with GAM to forma stable soluble MAb-GAM complex. One line of a 1.0 mg/ml MAb1-GAMcomplex was applied to (sprayed on) the nitrocellulose membrane(capture/control zone) to produce a total applied complex of 1 μg/linearcentimeter.

[0150] Four lines of a 220 μg/ml solution of the MAb1-GAM complex wereapplied to (sprayed on) the nitrocellulose membrane (test/read zone) toproduce a total applied complex of 22 ng/linear centimeter. The membranewas dried for 10 minutes at 37° C., then blocked with a stabilizedbuffer as described in Example 1, and dried again for 30 minutes at 37°C.

[0151] A polyester pad, pretreated with surfactant as described inExample 1, was laminated onto the lower portion of the strip to act as asample wick. A cellulose pad was laminated to the upper portion of thestrip to act as absorbent reservoir.

[0152] The completed card was cut into approximately 4 mm strips. Stripswere inserted into plastic housings with a hole/well located in thesample application area and a window in the test reading area.

Test Procedure

[0153] A 20 μl aliquot of cotinine test solution was added to the samplewell of each test strip device and allowed to absorb to the membrane for10-30 seconds. Thereafter, 20 μl of cotinine-tracer conjugate was addedto the sample well of each test strip device. The test strips wereallowed to develop for 10-15 minutes, then read visually.

Results

[0154] The results are exemplified in FIG. 6 and summarized below:

[0155] 0 ng cotinine/ml: control line visible, no test line visible;

[0156] 20 ng cotinine/ml: control line visible, one test line visible;

[0157] 200 ng cotinine/ml: control line visible, three test linesvisible; and

[0158] 500 ng cotinine/ml: control line visible, four test linesvisible.

Example 3 Cotinine Test with Analyte-Tracer Conjugate on the Glass-FiberPad Reagents

[0159] Cotinine-tracer conjugate, goat-anti mouse IgG antibody (GAM),anti-cotinine antibody (MAb1) and cotinine test solutions were preparedas described in Example 2.

Construction of Test Strips

[0160] A 25.4 mm×300 mm strip of nitrocellulose was affixed to a 15 miladhesive coated vinyl backing, to provide support.

[0161] The cotinine antibody (MAb 1) was mixed 1:1 with goat-anti mouseIgG (GAM) to form stable soluble complexes at a final concentration of 1mg/ml. Two lines of the MAb1-GAM complex were applied to thenitrocellulose membrane; a total of 1 μg/linear centimeter was appliedto each line. The membrane was dried for 15 minutes at 37° C.

[0162] Cotinine-tracer conjugate was diluted to 1.5 mg/ml with aconjugate buffer containing 10 mm Phosphate pH 7.4, 0.1% BSA, 0.05%Tween, 10 mm EDTA, 0.1% sodium azide and 20% sucrose. Four lines of thediluted cotinine-tracer conjugate solution were applied 1 mm apart tothe glass fiber pad (approximately 21 cm from the bottom of the strip)at 1.5 μg/linear centimeter for each line. The application of the fourlines was repeated and the pad dried for 1 hour at 37° C. in a dryingoven.

[0163] The dried glass fiber pad was laminated onto the lower portion ofthe strip to act as a sample wick. An untreated cellulose pad waslaminated to the upper portion of the strip to act as an absorbentreservoir.

[0164] The completed assay strips were cut to 4 mm in width and storedin a sealed foil pouch with approximately 1 gram of clay desiccant untilused.

Test Procedure

[0165] Three test strips were removed from the foil pouch and placed ona flat surface. A 100 μl test sample (containing 0, 20 or 200 ngcotinine/mi buffer) was applied to the sample application zone of eachtest strip. The strips were allowed to develop for 20 minutes and werethen read visually.

Results

[0166] The visual reading results are summarized below:

[0167] 0 ng cotinine/ml: strong control line visible, no test linevisible;

[0168] 20 ng cotinine/ml: strong control line visible, weak test linevisible; and

[0169]200 ng cotinine/ml: strong control line visible, strong test linewith intensity equal to the control line.

Example 4 Cotinine Test with Analyte-Tracer Conjugate on theNitrocellulose Reagents

[0170] Cotinine-tracer conjugate was prepared as described in Example 2.Goat-anti mouse IgG antibody (GAM), anti-cotinine antibody (MAb1) andcotinine test solutions were prepared as described in Example 2.

Test Strips

[0171] The cotinine antibody (MAb1)/goat-anti mouse IgG antibody (GAM)solution was applied to a strip of vinyl-backed nitrocellulose and driedas described in Example 3. Cotinine-tracer conjugate was diluted with aconjugate buffer as described in Example 3. The tracer conjugate wasdiluted to 750 μg/ml and applied to the nitrocellulose membrane at 0.75μg/linear centimeter. A total of four lines were applied 1 mm apart,beginning at the base (approximately 26 mm from the bottom of thestrip). The strip was dried for 1 hour at 37° C. in a drying oven.

[0172] An untreated cellulose pad was laminated to the upper portion ofthe strip to act as an absorbent reservoir. A 26 mm×300 mm strip of“Accuflow G” glass fiber material (Schleicher and Schuell, Inc., KeeneN.H.) for use as a sample pad was placed overlapping the top conjugateline to facilitate slow release of the cotinine-tracer conjugate.

[0173] The prepared test strips were cut to 4 mm widths and stored in asealed foil pouch with approximately 1 gram of clay desiccant untilused.

Test Procedure

[0174] Test strips were removed from the foil pouch and placed on a flatsurface. A 90 μl test sample (containing 0, 20 or 200 ng cotinine/mlbuffer) was applied to the sample application zone of each test strip.The strips were allowed to develop for 20 minutes and then readvisually.

Results

[0175] The strip with the sample pad overlapping the highestcotinine-tracer conjugate line gave the following visual results:

[0176] 0 ng cotinine/ml: strong control line, no test line;

[0177] 20 ng cotinine/ml: strong control line, detectable test line; and

[0178] 200 ng cotinine/ml: strong control line, strong test line withintensity equal to the control line.

Example 5 Methamphetamine Test With Analyte-Tracer Conjugate on theNitrocellulose Reagents

[0179] A methamphetamine-tracer conjugate was prepared methods similarto those described in Example 1 using methamphetamine-BSA conjugate(Arista Biological, Inc., Bethlehem, Pa.) instead of BSA-cotinine.Goat-anti mouse IgG antibody (GAM) was also obtained from AristaBiologicals. Methamphetamine monoclonal antibody was obtained from OmegaBiological, Inc (Bozeman, Mont.; Cat. #100-11-183, Clone Met 2).Methampetamine HCl (Sigma-Aldrich, St. Louis, Mo.) was used to preparetest solutions of 0, 10, and 100 ng/ml in saliva matrix (as described inExample 1).

Test Strips

[0180] One line of a 1.0 mg/ml solution of methamphetamineantibody/goat-anti mouse IgG antibody (GAM) complex was applied to astrip of vinyl-backed nitrocellulose membrane (capture/control line) toproduce a total applied complex of 1 μg/linear centimeter. Likewise, oneline of a 1.0 mg/ml solution of methamphetamine antibody/goat-anti mouseIgG antibody (GAM) complex was applied to a strip of vinyl-backednitrocellulose membrane (test/read line) to produce a total appliedcomplex of 1 μg/linear centimeter. The strip was then dried as describedin example 1.

[0181] Methamphetamine-tracer conjugate was diluted to 750 μg/ml with aconjugate buffer as described in Example 3, then applied to thenitrocellulose membrane at 0.75 μg/linear centimeter. A total of fourlines were applied 1 mm apart, beginning at the base (approximately 26mm from the bottom of the strip). The strip was dried for 1 hour at 37°C. in a drying oven.

[0182] An untreated cellulose pad was laminated to the upper portion ofthe strip to act as an absorbent reservoir. A 26 mm×300 mm strip of“Accuflow G” glass fiber material, for use as a sample pad, was placedoverlapping the top conjugate line to facilitate slow release of themethamphetamine-tracer conjugate.

[0183] The prepared test strips were cut to 4 mm widths and stored in asealed foil pouch with 1 gram of clay desiccant until used.

Test Procedure

[0184] Test strips were removed from the foil pouch and placed on a flatsurface. A 90 μl aliquot of a test sample (0, 10, 100 ng methamphetamineHCl/ml buffer) was applied to the sample application zone of each teststrip. The strips were allowed to develop for 20 minutes and then readvisually.

Results

[0185] The strips gave the following visual results:

[0186] 0 ng methamphetamine HCl/ml: strong control line, no test line;

[0187] 10 ng methamphetamine HCl/ml: strong control line, detectabletest line; and

[0188] 100 ng methamphetamine HCl/ml: strong control line, strong testline with intensity equal to the control line

Example 6 Preparation of Antibodies Immunogen Preparation

[0189] A bovine serum albumin (BSA)-cotinine conjugate was preparedusing cotinine trans-4-carboxylic acid as follows: To 0.25 ml of amixture of dimethylformamide and pyridine (1/1 by volume) was added 5 mgof trans-4-carboxycotinine (Sigma-Aldrich, St. Louis, Mo.), 5 mg ofN-hydroxysuccinimide and 15 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Sigma-Aldrich, St. Louis, Mo.). The mixturewas rotated to dissolve, and then left rotating for one hour to form theactive ester as described previously (Fjrowell and Landon, Ann ClinBiochem, 23:596-602, 1986). A 50 μl aliquot of this active ester wasadded to 1 ml of a 10 mg/ml BSA solution in 0.1 M phosphate buffer (pH7.4). The reaction mixture was rotated at room temperature for fourhours, then dialyzed against 10 mM phosphate buffer (pH 7.4).

[0190] The BSA-cotinine conjugate was diluted to a concentration of 5mg/ml bovine serum albumin in 10 mM phosphate buffer (pH 7.4). TheBSA-cotinine conjugate was stored aseptically in sterile 10 mM phosphatebuffer (pH 7.4) at 4° C. One (1.0) mg of this material was supplied forimmunizations. In addition, 1.0 mg of free cotinine (Sigma-Aldrich,product #C 5923) was supplied for booster immunizations.

Immunization Protocol

[0191] Four Balb/c female mice, age five weeks, were immunized using thefollowing procedure. Immunogen (BSA-cotinine, as prepared above) at 1mg/ml was mixed with an equal volume of RIBI adjuvant (Corixa, Seattle,Wash.). Each mouse received 20 μg total protein per subcutaneousinjection on days 0, 7 and 21. Retro-orbital collection of blood wasperformed on day 35. Serum from each mouse was extracted by centrifugingat 3000×g for ten minutes, and titers of each serum were measured usinga sandwich ELISA method. After resting for ˜30 days, the mouse with thestrongest anti-cotinine titer was boosted with an intra-peritonealinjection of the BSA-cotinine conjugate (50 μg prepared in RIBIadjuvant) and an intravenous injection of free cotinine (5 μg in 0.1 Mphosphate buffered saline, pH 7.4). Fusion of splenocytes from the mouseto NSO cells (Balb/c myeloma, non-secreting) was performed four daysafter the boost. Mother clones (polyclonals) were initially screenedusing the ELISA method described below. Positive mother clones werefurther sub-cloned. Monoclones were isolated using limiting dilution.

Antibody Selection

[0192] An initial screen was performed using sandwich ELISA.High-binding 96-well microtiter plates (Nalge Nunc, Rocherster, N.Y.)were coated overnight at 4° C. with 1 μg/well of BSA-cotinine diluted in10 mM phosphate buffered (pH 7.4) saline (PBS). The coating solution wasremoved by inversion of the plate. Cell supernatant (100 μl) was addedto each well and incubated for one hour at 37° C. The plate was washedfive times with PBS containing 0.05% Tween 20. Goat anti-mouse heavy andlight chain antibody (GAM) conjugated to horseradish peroxidase (HRP)(Jackson ImmunoResearch Labs, West Grove, Pa.) was diluted 1:5000 inPBS-Tween. A 100 μl aliquot of the diluted HRP preparation was added toeach well and incubated for 30 minutes at 37° C. Positive wells werevisualized by adding 100 μl of One-step TMB substrate (Kirkegaard &Perry Laboratories, Gaithersburg, Md.) to each well, followed byincubation for 15 minutes at room temperature. A second ELISA screen(for specificity) was performed on initial positives using BSA-coatedwells. The final ELISA assay was performed using wells coated withtwo-fold dilutions of immunogen from 100 ng/well to 1 ng/well, keepingantibody concentration constant.

[0193] All ELISA assays used cell supernatants from cultures atmid-growth phase, and at 1:5 or 1:10 dilutions. Approximately 10 ml ofsupernatant from each of the highest titered clones was stored at −20°C. for later testing.

[0194] Selection of the anti-cotinine clones for differential affinitieswas performed using a lateral flow immunochromatographic assay.Immunochromatographic test strips were prepared as described inExample 1. The primary capture zone consisting of two transverselyoriented lines of goat anti-mouse (GAM-IgG) IgG antibody (1 μg/μl inPBS) applied beginning approximately 3 mm from the origin of thenitrocellulose. The two lines were placed 1 mm apart to produce a totalapplied complex of 1 μg/linear centimeter. The secondary capture zoneconsisted of one line of streptavidin (2 mg/ml) applied approximately 5mm from the second line of the primary capture zone to produce a totalapplied complex of 0.5 μg/linear centimeter.

[0195] After application of the primary and secondary capture molecules,the strips were allowed to dry, then blocked by immersion in a blockingbuffer (0.5% BSA, 4% sucrose 150 mM phosphate buffered saline, pH 7.4)for one minute, to prevent further protein adsorption. The strips werethen dried for ten minutes at 37° C.

[0196] Latex-BSA-cotinine-biotin conjugate was prepared as described inExample 1 (above).

[0197] The tissue culture supernatants were tested as follows:

[0198] 1) 25 μl of each supernatant was added to separate 12×75 mmborosilicate glass test tubes;

[0199] 2) either 125 μl of a solution of free cotinine (250 ng/ml) in abuffer (10 mM phosphate buffered saline, 0.05% Tween 20, 0.1% BSA, pH7.4) or 125 μl of buffer without cotinine, was added to each tube;

[0200] 3) 1.5 μl of latex-BSA-cotinine-biotin (10 mg/ml) was added toeach tube; and

[0201] 4) one test strip was added to each tube and allowed to run tocompletion (approximately 12 minutes).

[0202] The optimum clone for the primary antibody showed two distinctand important characteristics:

[0203] 1) In the tubes containing buffer alone (no free cotinine), 100%binding of the conjugate in the GAM-IgG primary capture zone, with thehighest percentage of conjugate bound by the first line within thatzone; and

[0204] 2) In the tubes containing the 25 ng of free cotinine,displacement of a relatively high percentage of latex conjugate,measured by binding in the secondary capture zone.

[0205] These binding characteristics are indicative of a similaraffinity of the clone for both free and bound cotinine.

[0206] The optimum clone for the secondary capture zone exhibited onlybinding in the primary capture zone, with the highest percentage boundby the first line within that zone, and did not exhibit any displacementby free cotinine. This type of binding characteristic indicates that theclone has an affinity for only the bound form of cotinine.

[0207] The optimum clone for primary capture was clone #5-3H, and theoptimum clone for secondary capture was clone #10-3A.

Antibody Production & Purification

[0208] Once optimum clones were selected, the appropriate cell lineswere frozen and stored in liquid nitrogen. Growth of the hybridoma celllines was initiated from frozen cells in 24-well tissue culture platesin a hybridoma growth medium consisting of DMEM (high glucose)/10%FetalClone® 1 serum (HyClone, Logan, Utah). Cells were maintained at 2to 7×100,000 cells/ml by expansion into fresh medium twice weekly. ACELLine™CL 1000 (Integra Biosciences, Jamesville, Md.) was used forantibody production (see manufacturers instructions) with up to 10⁹cells in less than 12 ml of hybridoma growth medium in the innerchamber. The outer chamber contained 950 ml of serum-free DMEM.Approximately ⅓ to ⅔ of outer chamber was collected twice weekly,followed by volume replacement with fresh medium.

[0209] Purification of the monoclonal antibodies was achieved directlyfrom the growth medium over a column with recombinant Protein Aimmobilized on porous glass beads (ProSep A; Pierce Chemical Co.,Rockford, Ill.), following the manufacturers instructions. Immediatelyfollowing purification, the monoclonal antibodies were dialyzed againstone liter of 10 mM phosphate buffered saline (0.05% sodium azide, pH7.4) in Spectra/Por® 7, 10,000 MW cut-off membrane (SpectrumLaboratories, Rancho Dominguez, Calif.) for 24 hours with at least twobuffer changes.

[0210] In view of the many possible embodiments to which the principlesof our invention may be applied, it should be recognized that theillustrated embodiments are only examples of the invention and shouldnot be taken as limitations on the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A device for determining presence and/or amount of ananalyte in a fluid sample comprising: a mobilization zone comprising amobile or mobilizable detectable tracer molecule; a sample applicationarea; a primary capture area comprising a first immobilized bindingpartner having a binding affinity for the analyte and a binding affinityfor the detectable tracer molecule; and a secondary capture areacomprising a second immobilized binding partner having a bindingaffinity for the analyte and a binding affinity for the detectabletracer molecule, wherein the sample application area, primary capturearea and secondary capture area are in fluid continuous contact, and thefirst immobilized binding partner has an equal or a lower apparentaffinity for the analyte than it has for the detectable tracer molecule.2. The device of claim 1, wherein the detectable tracer molecule isassociated with the device in such a way that, during operation of thedevice, it contacts the primary capture area after a sample contacts theprimary capture area.
 3. The device of claim 1, wherein, duringoperation of the device, the detectable tracer molecule migrates throughthe device at a rate slower than a rate at which the analyte in a samplemigrates through the device.
 4. The device of claim 3, wherein slowermigration of the tracer molecule is caused by a molecular weight of thetracer molecule.
 5. The device of claim 3, wherein slower migration ofthe tracer molecule is caused by a physical or temporal placement of thetracer molecule on the device.
 6. The device of claim 5, wherein thetracer molecule is placed on the device after a sample is placed on thedevice.
 7. The device of claim 1 further comprising at least one filterpad in a path of flow of the fluid.
 8. The device of claim 5, whereinthe filter pad is pretreated with at least one reagent to enhance thesensitivity of the assay device.
 9. The device of claim 6 wherein the atleast one reagent is selected from the group consisting of buffers,detergents, and anticoagulants.
 10. The device of claim 1 wherein thefirst and second immobilized binding partners are selected from thegroup consisting of antibodies, antigens and haptens.
 11. The device ofclaim 1 wherein the first and second binding agents for the analyte areidentical.
 12. The device of claim 1 wherein the first and secondbinding agents are each anti-analyte antibodies.
 13. The device of claim1, wherein the tracer molecule comprises an analyte molecule or ananalyte analog molecule.
 14. The device of claim 1, wherein the tracermolecule comprises a visually detectable label.
 15. The device of claim1, wherein the analyte is selected from the group consisting of antigensof infectious diseases, antibodies to antigens of infections diseases,hormones, growth factors, therapeutic drugs, drugs of abuse and productsof the metabolism of drugs of abuse, and haptens.
 16. The device ofclaim 15, wherein the analyte antibodies are selected from the groupconsisting of antibodies to HIV, antibodies to HTLV, antibodies toHelicobacter pylori, antibodies to hepatitis, antibodies to measles,antibodies to mumps, and antibodies to rubella.
 17. The device of claim15, wherein the therapeutic drugs and drugs of abuse or products of themetabolism of drugs of abuse are selected from the group consisting oftetrahydrocannabinol, nicotine, cotinine, ethanol, theophylline,phenytoin, acetaminophen, lithium, diazepam, nortryptyline,secobarbital, and phenobarbitol, methamphetamine and fragments,mimetics, analogs or derivatives thereof.
 18. The device of claim 17,wherein the analyte is a product of metabolism of a drug of abuse, andthe product of metabolism comprises cotinine.
 19. The device of claim15, wherein the hormones are selected from the group consisting oftestosterone, estradiol, estriol, 17-hydroxyprogesterone, progesterone,thyroxine, thyroid stimulating hormone, follicle stimulating hormone,and luteinizing hormone, and fragments, mimetics, analogs or derivativesthereof.
 20. The device of claim 1, wherein the quantity of the secondspecific binding partner in the secondary capture area is such that thequantity of tracer molecule binding to the secondary capture area, andby correlation the amount of the analyte in a tested sample, isindicated by the intensity of detection signal of the tracer molecule inthe secondary capture area.
 21. The device of claim 1 wherein the areaof the secondary specific binding partner immobilized on thechromatographic medium is divided into at least two discrete andnon-overlapping bands, with the quantity of the second specific bindingpartner in each band being such that the quantity of tracer moleculebinding to the secondary capture area, and by correlation the amount ofthe analyte in a tested sample, is indicated by the number of bands towhich the tracer molecule binds.
 22. A device for determining presenceand/or amount of an analyte in a fluid sample comprising: a mobilizationzone comprising a mobile or mobilizable detectable tracer molecule; asample application area; a primary capture area comprising a firstimmobilized binding partner having a binding affinity for the analyteand a binding affinity for the detectable tracer molecule; and asecondary capture area comprising a second immobilized binding partnerhaving a binding affinity for the analyte and a binding affinity for thedetectable tracer molecule, wherein, during operation of the device, thedetectable tracer molecule contacts the primary capture area after thesample contacts the primary capture area.
 23. A method for detectingand/or quantitating an analyte in a fluid sample, comprising: applying aliquid sample to a substrate along which the samples migratessequentially to a primary capture area and a secondary capture area,wherein the primary capture area binds the analyte with an equal or alower apparent affinity than it binds a detectable tracer molecule; andthe secondary capture area binds the detectable tracer molecule withhigh affinity; and reading a detectable signal from bound detectabletracer molecule in the secondary capture area, wherein the detectablesignal indicates the presence of analyte in the sample.
 24. The methodof claim 23, further comprising: applying a detectable tracer moleculeto the substrate.
 25. The method of claim 24, wherein the detectabletracer molecule is applied to the substrate before the sample.
 26. Themethod of claim 24, wherein the detectable tracer molecule is applied tothe substrate after the sample.
 27. The method of claim 24, wherein thedetectable tracer molecule and the sample are applied simultaneously.28. The method of claim 23, wherein the detectable signal has anintensity, and the intensity of the signal correlates with the amount ofanalyte in the sample.
 29. The method of claim 23, wherein the first andsecond binding partners are immobilized on the substrate.
 30. The methodof claim 23, using the device of claim
 1. 31. A method for detectingand/or quantitating an analyte in a fluid sample, comprising: contactingthe fluid sample with the device of claim
 1. 32. The method of claim 31,comprising: applying the sample to the sample application area of thedevice; allowing the sample to migrate along the device; and allowingthe detectable tracer molecule to migrate along the device, wherein adetectable signal from bound detectable tracer molecule in the secondarycapture area indicates the presence of the analyte in the sample. 33.The method of claim 31, wherein the mobile detectable analyte analog isapplied to the device no earlier than the sample is applied to thedevice.
 34. The method of claim 33, wherein the analyte-tracer conjugateis mixed with the sample prior to application to the sample applicationarea.
 35. The method of claim 31, further comprising quantifying theamount of analyte in the sample, wherein the amount of analyte in thesample is proportional to the signal in the second capture area.
 36. Themethod of claim 31, wherein the sample migrates along the test stripdevice by capillary action.
 37. The method of claim 31, wherein theanalyte has a molecular weight of about 100-1,000 Daltons.
 38. Themethod of claim 31, wherein the analyte has a molecular weight ofgreater than 1,000 Daltons.
 39. The method of claim 31, wherein thesample is a fluid sample.
 40. The method of claim 39, wherein the fluidsample is selected from the group consisting of urine, blood, tears,sweat and saliva.
 41. The method of claim 40, wherein the fluid sampleis saliva.
 42. The method of claim 41, further comprising providing anoral fluid sample combined with a bile acid bile or salt in aconcentration sufficient to reduce occurrence of false positives in theimmunoassay.
 43. The method of claim 42, wherein the bile acid or bilesalt ranges in concentration from about 0.1 weight percent to about 1.0weight percent of the oral fluid/bile salt or bile acid combination. 44.The method of claim 43, further comprising contacting a chelator ofdivalent cations with the oral fluid sample.
 45. A test kit for thedetection and/or the determination of an analyte in a sample comprising:(a) the chromatographic assay device of claim 1; and (b) instructions.46. The kit of claim 45, further comprising an aliquot of analyte-tracerconjugate.